The ability of platelets to regulate thrombin generation at their membrane surface is central to their role in hemostasis, thrombosis, and atherosclerosis. Thrombin generation is effected through the assembly and function of the enzymatic complex, and atherosclerosis. Thrombin generation is effected through the assembly and function of the enzymatic complex, Prothrombinase, consisting of a I.I stoichiometric Ca2+- dependent complex of the cofactor factor Va and the serine protease factor Xa. Subsequent to platelet activation, release platelet factor Va and/or plasma-derived factor Va bind to the platelet membrane surface and in so doing form at least part of the receptor for factor Xa. Thus, several protein/protein and protein/membrane interactions participate in and regulate complex assembly. One goal of this project is to define how platelets actively participate in and regulate Prothrombinase assembly and function. The following hypotheses have been formulated and will be tested. Platelets regulate thrombin generation through 1) the expression of two discrete platelet subpopulations-both bind factor Va, only one binds both factors Va and Xa; and 3) through the agonist-induced release of platelet factor Va which is resistant to inactivation by activation by activated protein C and plasmin, and functionally and physically unique when compared to plasma-derived factor Va. Effort will be placed on isolating and characterizing these discrete platelet populations, isolating and/or expression cloning the platelet membrane receptors for factor Va and Xa, and characterizing platelet-derived factor Va, functionally and physically as related to its participation in Prothrombinase. Because thrombin once formed positively regulates Prothrombinase assembly and function through platelet activation and release of platelet factor Va, a second goal is to define how thrombin interacts through platelet activation and release of platelet factor Va, a second goal is to define how thrombin interacts with platelet membrane proteins to modulate its activity. Effort will be place don testing the hypothesis that the platelet high affinity binding site for thrombin is 1) a unique platelet membrane protein that resembles hirudin and renders the thrombin molecule transiently inactive and, 2) is distinct from glycoprotein 1b. This will be accomplished in part through expression cloning of this unique platelet membrane protein and demonstration that anti-glycoprotein 1b antibodies shown to inhibit thrombin-induced platelet activation do so through their cross reactivity with PAR1. Successful completion of these goals will demonstrate the mechanisms by which platelets actively regulate both the generation and function of thrombin at their membrane surface.